Effects of Single and Double Mutations in Plastocyanin on the Rate Constant and Activation Parameters for the Rearrangement Gating the Electron-Transfer Reaction between the Triplet State of Zinc Cytochromecand Cupriplastocyanin

Abstract

The unimolecular rate constant for the photoinduced electron-transfer reaction 3Zncyt/pc(II) --> Zncyt+/pc(I) within the electrostatic complex of zinc cytochrome c and spinach cupriplastocyanin is kF. We report the effects on kF of the following factors, all at pH 7.0: 12 single mutations on the plastocyanin surface (Leu12Asn, Leu12Glu, Leu12Lys, Asp42Asn, Asp42Lys, Glu43Asn, Glu59Gln, Glu59Lys, Glu60Gln, Glu60Lys, Gln88Glu, and Gln88Lys), the double mutation Glu59Lys/Glu60Gln, temperature (in the range 273.3-302.9 K), and solution viscosity (in the range 1. 00-116.0 cP) at 283.2 and 293.2 K. We also report the effects of the plastocyanin mutations on the association constant (Ka) and the corresponding free energy of association (DeltaGa) with zinc cytochrome c at 298.2 K. Dependence of kF on temperature yielded the activation parameters DeltaH, DeltaS, and DeltaG. Dependence of kF on solution viscosity yielded the protein friction and confirmed the DeltaG values determined from the temperature dependence. The aforementioned intracomplex reaction is not a simple electron-transfer reaction because donor-acceptor electronic coupling (HAB) and reorganizational energy (lambda), obtained by fitting of the temperature dependence of kF to the Marcus equation, deviate from the expectations based on precedents and because kF greatly depends on viscosity. This last dependence and the fact that certain mutations affect Ka but not kF are two lines of evidence against the mechanism in which the electron-transfer step is coupled with the faster, but thermodynamically unfavorable, rearrangement step. The electron-transfer reaction is gated by the slower, and thus rate determining, structural rearrangement of the diprotein complex; the rate constant kF corresponds to this rearrangement. Isokinetic correlation of DeltaH and DeltaS parameters and Coulombic energies of the various configurations of the Zncyt/pc(II) complex consistently show that the rearrangement is a facile configurational fluctuation of the associated proteins, qualitatively the same process regardless of the mutations in plastocyanin. Correlation of kF with the orientation of the cupriplastocyanin dipole moment indicates that the reactive configuration of the diprotein complex involves the area near the residue 59, between the upper acidic cluster and the hydrophobic patch. Kinetic effects and noneffects of plastocyanin mutations show that the rearrangement from the initial (docking) configuration, which involves both acidic clusters, to the reactive configuration does not involve the lower acidic cluster and the hydrophobic patch but involves the upper acidic cluster and the area near the residue 88.

A Single Arginyl Residue in Plastocyanin and in Cytochrome c6 from the Cyanobacterium Anabaenasp. PCC 7119 Is Required for Efficient Reduction of Photosystem I

“…These findings allow us to conclude that Arg-88 is a crucial residue not only for long range electrostatic attractions between Pc and PSI but also for electron transfer. It should be noted that glutamine at position 88 of spinach Pc has been replaced with asparagine, glutamate, lysine, and tyrosine with no significant changes in the kinetics of PSI reduction (27,28).…”

Dissecting regulation mechanism of the FMN to heme interdomain electron transfer in nitric oxide synthases

“…In the cases of true and coupled ET, the observed ET rates are expected to be independent of viscosity [117]. A recent electron transfer kinetics study at various temperatures suggested that the FMN–heme IET in nNOS holoenzyme may be coupled to the pre-requisite conformational movement of the FMN domain [118].…”

High‐Pressure Probing of a Changeover in the Charge‐Transfer Mechanism for Intact Cytochrome c at Gold/Self‐Assembled Monolayer Junctions

“…[4][5][6] However, even for small and well-characterized proteins such as cytochrome c (CytC), [7,8] an intrinsic CT mechanism in "homogeneous" systems is difficult to recognize conclusively as a consequence of the extra structural and dynamic environmental complexity introduced by the participating redox partner. [4,5,[9][10][11] The covalent attachment of "small" complex ions as the redox counterparts at different external sites of CytC [12,13] does not warrant sufficiently smooth variation of intrinsic parameters, such as an electronic coupling (correlated with the CT distance; see below), because of the highly inhomogeneous nature of the protein interior. In this context, artificial bioelectrochemical devices made of electrode-deposited self-assembled monolayer (SAM) films of variable composition and thickness, and CytC or other redox proteins attached or freely diffusing to the SAM terminal groups (also subject to wide variations), were proven to be systems with wellcontrolled variable parameters, and hence suitable for fundamental studies [8,[14][15][16][17][18] and some technological applications.…”

“…[18] Somewhat larger slopes were found for an ET within the "homogeneous" system involving zinc-substituted CytC, and wild-type and mutant cupriplastocyanin, which ranged from 0.7 to 0.9. [9][10][11] Interestingly, full viscosity control (d % 1) has been observed for photoinduced CT from the artificial Rucoordinated polypeptide electron donor to ferri-CytC, [48] which occurs through the loosely bound (encounter) complex as opposed to the "preformed" (tight) complex involving the same reactants (d % 0.6); both patterns occur simultaneously. The latter findings closely match the whole pattern of CytC bioelectrochemical CT in both the tightly bound and freely diffusing (to Au-deposited SAMs comprising w-COOH and w-OH) regimes, respectively.…”